EP2951298A1 - Procédés et moyens pour augmenter la tolérance aux contraintes et la biomasse dans des plantes - Google Patents

Procédés et moyens pour augmenter la tolérance aux contraintes et la biomasse dans des plantes

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Publication number
EP2951298A1
EP2951298A1 EP14701975.6A EP14701975A EP2951298A1 EP 2951298 A1 EP2951298 A1 EP 2951298A1 EP 14701975 A EP14701975 A EP 14701975A EP 2951298 A1 EP2951298 A1 EP 2951298A1
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EP
European Patent Office
Prior art keywords
seq
plant
hdc1
protein
plants
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP14701975.6A
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German (de)
English (en)
Inventor
Anna Amtmann
Matthew Hannah
Veronique Gossele
Manuel LOPEZ-VERNAZA
Giorgio PERRELLA
Christoph VERDUYN
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University of Glasgow
Bayer CropScience LP
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University of Glasgow
Bayer CropScience LP
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Priority to EP14701975.6A priority Critical patent/EP2951298A1/fr
Publication of EP2951298A1 publication Critical patent/EP2951298A1/fr
Withdrawn legal-status Critical Current

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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8267—Seed dormancy, germination or sprouting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01098—Histone deacetylase (3.5.1.98), i.e. sirtuin deacetylase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates generally to the field of plant molecular biology and concerns a method for improving plant tolerance to stress conditions. More specifically, the present invention concerns a method for increasing stress tolerance and growth and for reducing ABA sensitivity, comprising increasing the expression and/or activity of a H ISTONE DEACETYLASE COMPLEX 1 (HDC1 ) protein in a plant.
  • the present invention also concerns plants having an increased expression and/or activity of HDC1 , which plants have inter alia an increased stress tolerance, biomass, yield and reduced ABA sensitivity relative to corresponding wild-type plants.
  • the invention also provides chimeric genes, nucleic acids and polypeptides encoding such HDC1 proteins.
  • ABA abscisic aid
  • ABA induces the closure of stomatal pores to minimise transpirational water loss and initiates the production of proteins and metabolites that prevent cellular damage during drying, thawing and osmotic shock.
  • Cross-talk between ABA and other hormones such as ethylene (ET), gibberellin (GA), cytokinin (CK) and jasmonic acid (JA) integrates physiological and metabolic responses with plant growth and development (Chinnusamy et al., 2004, Journal of Experimental Botany 55, 225-236; Achard et al., 2006, Science 31 1 , 91 -94; Daszkowska-Golec, 201 1 , Omics 15, 763-774; Wilkinson et al., 2012, Journal of Experimental Botany 63, 3499- 3509).
  • chromatin remodelling has emerged as an important factor for transcriptional responses to ABA (Chinnusamy et al., 2008, J Integr Plant Biol 50, 1 187-1195).
  • nucleosome assembly proteins and subunits of SWI/SNF chromatin-remodeling complexes have been reported to alter ABA sensitivity (Saez et al., 2008, Plant Cell 20, 2972-2988; Liu et al., 2009, Mol Plant 2, 688-699).
  • Histone deacetylation (HD) has emerged as an important regulatory process during environmental stress (Kim et al. 2012, Plant Cell Physiol 53: 797-800).
  • Histone de-acetylases remove active acetylation marks from lysine residues of histones 3 and 4 which in turn leads to repression of gene transcription both through interaction with gene-specific repressors and through general chromatin compression (Kurdistani and Grunstein, 2003, Nat Rev Mol Cell Bio 4, 276-284).
  • HDACs belong to three different structural groups; Type-I HDACs, similar to Rpd3/HDAC1 -type enzymes in yeast and animals, Sirtuins, homologous to similar enzymes in other eukaryotes, and HD-tuins, a plants specific class of proteins (Pandey et al.
  • seedlings of hd2c knockout mutants are ABA-hypersensitive as are seedlings of knockdown lines (axe1-5, CS2483) for HDA6, a Rpd3/HD1 -type HDAC (Sridha and Wu, 2006, supra; Luo et al., 2012, Journal of Experimental Botany 63, 3297-3306, Chen et al. 2010, Exp Bot 61 : 3345-3353). It was further shown that HD2C interacts with HDA6, and that crossing of axef-5with rtc 2c further increases ABA-sensitivity of seedlings (Luo et al., 2012, supra).
  • histone Rpd3/HD1 -type histone de-acetylases act in conjunction with gene-specific transcriptional repressors (e.g. Ume6), a co-repressor (Sin3), Sin3-associated peptides (e.g.SAP18), histone-binding proteins (e.g. Ume1 , RbAp46/48, TBL1 ) as well as functionally uncharacterised proteins (e.g. Rxt1 -3) (Carrozza et al., 2005, Bba-Gene Struct Expr 1731 , 77-87; Chen et al. 2012, Curr Biol 22: 56-63; Roguev and Krogan, 2007, Nat.
  • gene-specific transcriptional repressors e.g. Ume6
  • Sin3 co-repressor
  • Sin3-associated peptides e.g.SAP18
  • histone-binding proteins e.g. Ume1 , RbAp46
  • yeasts assemble a large and a small Sin3 complex (Rpd3L/S in S. cerevisiae, l/l I in S. pombe (Roguev and Krogan, 2007, supra) while mammals and insects assemble at least three distinct complexes (Mi-2/NuRD, CoREST and N-CoR/SMRT (Yang and Seto, 2008, supra).
  • Knockout/knockdown of these genes in A. thaliana caused similar phenotypes as knockdown of HDA6, e.g. ABA-hypersensitivity and delayed flowering (Song et al., 2005,supra; Song and Galbraith, 2006,supra).
  • knockout of an A. thaliana homologue of mammalian TBL1 did not alter ABA-sensitivity but caused hypersensitivity of seedlings to cold (Zhu et al., 2008, Proc. Natl. Acad. Aci. USA 105, 4945-4950).
  • WO04/022735 discloses proteins OsHDACI , OsHDAC2 and OsHDAC3, which function as histone deacetylase, a gene coding for said proteins, and a method for producing a plant having a high growth rate by expressing said gene in the plant.
  • Jang et al. discloses that, while constitutive over-expression of HDAC1 -3 in rice resulted in calli which could not be propagated, inducible overexpression also caused developmental aberrations in addition to enhanced growth.
  • WO04/035798 discloses a method for altering characteristics of a plant and describes the identification of genes that are upregulated or downregulated in transgenic plants overexpressing E2Fa/DPa and the use of such sequences to alter plant characteristics.
  • the present invention provides a contribution over the art by disclosing a new HDAC-interacting protein that can be used to modulate plant stress response, ABA-sensitivity, growth and flowering.
  • the invention provides a method for increasing tolerance of a plant, plant part, plant organ or plant cell to stress conditions, preferably mild or moderate stress conditions; or for reducing ABA sensitivity of a plant, plant part, plant organ or plant cell; and/or for increasing biomass and/or yield and/or growth rate of a plant, plant organ or plant part; and/or for accelerating flowering time of a plant; comprising the step of
  • Said increasing the expression and/or activity of a protein having the activity of the protein with the amino acid sequence of SEQ ID NO. 6 may comprise expressing in said plant cell, plant part, plant organ or plant a chimeric gene comprising the following operably linked elements:
  • a nucleic acid which when transcribed results in an increased activity and/or expression of a protein having the activity of the protein encoded by SEQ ID NO. 6
  • the nucleic acid encodes a protein having the activity of the protein with the amino acid sequence of SEQ ID NO. 6, or the nucleic acid comprises a nucleic acid sequence encoding a protein having at least 70% sequence identity to SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40 or SEQ ID NO.
  • the nucleic acid comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 1 1 , SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21 , SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 , SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37 or SEQ ID NO. 39.
  • the promoter may be a constitutive promoter or an inducible promoter.
  • the plant is selected from wheat, oilseed rape, lettuce, tobacco, cotton, corn, rice, vegetable plants, carrot, cucumber, leek, pea, melon, potato, tomato, sorghum, rye, oat, sugarcane, peanut, flax, bean, sugar beets, soy bean, sunflower and ornamental plants.
  • the stress condition can be selected from drought stress, salt stress, low nutrient levels, high light stress and oxidative stress.
  • the invention furthermore provides a method for enhancing survival of a plant, plant part, plant organ or plant cell under severe stress conditions, or for enhancing recovery after severe stress of a plant, plant part, plant organ or plant cell, or for delaying the flowering time of a plant, comprising the step of:
  • the reducing the expression and/or activity may comprise expressing in said plant cell, plant part, plant organ or plant a chimeric gene comprising the following operably linked elements:
  • a nucleic acid which when transcribed results in a decreased activity and/or expression of a protein having the activity of the protein encoded by SEQ ID NO. 6
  • the nucleic acid may when transcribed yield an HDC1 inhibitory RNA molecule.
  • the promoter is an inducible promoter.
  • the invention also provides a chimeric gene as described above.
  • a plant, plant part, plant organ, plant cell or seed that has been modified according to the invention so as to have an increased or reduced expression and/or activity of a protein having the activity of the protein with the amino acid sequence of SEQ ID NO. 6. when compared to a control plant, such as a plant, plant part, plant organ, plant cell or seed comprising a chimeric gene according to the invention.
  • the plant, plant part, plant organ, plant cell or seed of the invention can be oilseed rape, lettuce, tobacco, cotton, com, rice, wheat, vegetable plants, carrot, cucumber, leek, pea, melon, potato, tomato, sorghum, rye, oat, sugarcane, peanut, flax, bean, sugar beets, soya, sunflower or ornamental plants.
  • a method for reducing yield penalty of a plant under stress conditions comprising increasing in said plant the expression and/or activity of a protein having the activity of the protein with the amino acid sequence of SEQ ID NO. 6., for example by expressing in said plant a chimeric gene as described above for increasing the activity and/or expression of a protein having the activity of the protein encoded by SEQ ID NO. 6 (i.e. the chimeric gene comprising a nucleic acid which when transcribed results in an increased activity and/or expression of a protein having the activity of the protein encoded by SEQ ID NO. 6 operably linked to a plant- expressible promoter and optionally a plant-functional a 3' end region).
  • a method for producing a plant with increased tolerance to stress conditions comprising the steps of:
  • the invention also provides a method for modulating histone acetylation in a cell, comprising the step of modulating the expression and/or activity of a protein having the activity of the protein encoded by SEQ ID NO. 6 in said cell, wherein increasing the expression and/or activity of said protein inhibits histone acetylation and decreasing the expression and/or activity of said protein enhances histone acetylation.
  • a chimeric gene as described above for increased activity and/or expression of a protein having the activity of the protein encoded by SEQ ID NO. 6 to increase the tolerance of a plant, plant part, plant organ or plant cell to (mild or moderate) stress conditions; or to reduce ABA sensitivity of a plant, plant part, plant organ or plant cell; or to increasing biomass or yield or growth rate of a plant, plant organ or plant part; or to accelerate flowering time of a plant.
  • the invention also provides the use of a plant which has been modified so as to have an increased expression and/or activity of a protein having the activity of the protein with the amino acid sequence of SEQ ID NO. 6., for instance of a plant comprising a chimeric gene as described above for increasing the activity and/or expression of a protein having the activity of the protein encoded by SEQ ID NO. 6, to produce a population of plants with increased tolerance to (mild or moderate) stress conditions, or with reduced ABA sensitivity, or with increased biomass or yield or growth rate, or with an accelerated flowering time.
  • the invention provides a protein having the activity of the protein with the amino acid sequence of SEQ ID NO. 6. That protein may have at least 70% sequence identity to SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40 or SEQ ID NO. 41 .
  • a nucleic acid encoding the above protein i.e. protein having the activity of the protein with the amino acid sequence of SEQ ID NO. 6, is also provided. That nucleic acid may have at least 70% sequence identity to SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 1 1 , SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21 , SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 , SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37 and SEQ ID NO. 39.
  • FIG. 1 HDC1 proteins have extended from ancestral Rxt3 proteins.
  • A Cluster dendrogram of predicted protein sequences of HDC1/Rxt3 genes in yeast, algae, protozoa, mosses and higher plants, based on alignment of predicted amino acid sequences provided in Supplemental File 1 .
  • B Schematic view of conserved and novel parts of higher plant HDC1 proteins. For the Rxt3 part of the protein an alignment of the A. thaliana (At) sequence with sequences from Brachypodium distachyon (Bd) HDC1 and yeast (Sc) Rxt3 to A. thaliana (At) is inserted.
  • a conserved Protein domain family signature 'histone de-ace tylation Rxt3' (PF08642) is marked with a box.
  • HDC1 is a ubiquitous nuclear protein.Tissue expression pattern and sub-cellular localization of HDC1.
  • GUS staining shows HDC1 promoter activity in A. thaliana seeds (A), root and shoot of seedlings (B) and mature plants (C), rosette leaves (D) and flower buds (E). No staining is visible inside anthers or stigmas (F, arrows).
  • FIG. 3 Co-localization of HDC1 with HDA6 and HDA19 within nuclei of transiently expressing tobacco epidermis cells.
  • Each row contains the following images from left to right: bright field, GFP fluorescence, RFP fluorescence, GFP/RFP overlay, quantitative comparison GFP and RFP signals along line scan (arrows in overlay images).
  • HDC1 co-localizes with HDA6 (A-C) and HDA19 (D-F) in the entire nucleus (A, D), in distinct speckles (B-E) or in the nucleolus (C, F). Scale bar is 10 pm.
  • Figure 4 HDC1 interacts with histone deacetylases HDA6 and HDA19 in a ratiometric BiFC assay.
  • A '2-in-1 ' vectors constructed for ratiometric BiFC assay containing N- and C-terminal halves of YFP (nYFP, cYFP) fused to HDC1 , HDA6, HDA19 and SIN3 as well as a full-length RFP.
  • HDC1 interacts with histone deacetylases in planta and facilitates H3K9/14 deacetylation.
  • A Anti-His Western blots of recombinant HDC1 -His after in-vitro pull-down with recombinant GST-HDA6 (second lane) and GST- HDA19 (third lane). The first lane contains a positive control (recombinant HDC1 -His), the last lane contains a negative control (pull down with GST alone).
  • B Anti-HDC1 Western blots of native HDC1 after pull-down from nuclei-enriched protein samples of A.
  • HDC1 thaliana wildtype (WT, left) or HDC1 knockout plants (hdd -1 , right) with recombinant GST-HDA6 (second lanes) or GST-HDA19 (third lanes).
  • HDC1 is recognized in the untreated protein samples from wildtype (input), and in wildtype samples after pull-down with GST-HDA6/19 but not with GST alone. HDC1 is not found in protein samples (input or pull-downs) from knockout plants.
  • the lower panel shows the membrane re-probed with anti-GST confirming presence of the bait.
  • Western blot with anti-H3K9K14ac shows increased amounts of acetylated H3K49K14 in protein extract from A.
  • H3K49K14Ac/H3 signal ratios in wildtype, hdd -1 and HDC1 c lines were determined after quantification of bands with Image J. Bars are means ⁇ SE from at least three Western blots. Asterisk indicates significant (p ⁇ 0.05) difference to WT and to HDC1 c.
  • FIG. 6 Confirmation of hdd -1 knockout and HDC1 over-expressing lines.
  • A Position of T-DNA and primer pairs in the genomic DNA of A. thaliana hdd -1 knockout line (GABI-Kat 054G03). Numbers indicate position of primer pairs used for genotyping.
  • B HDC1 mRNA in wildtype and hdd -1 as determined by semi-quantitative RT-PCR using the primer pairs indicated in A. Tubulin 9 (Tub 9) was used as a loading control.
  • C Western blot with anti-HDC1 detects HDC1 in A. thaliana wildtype but not in hdd -1 .
  • HDC1 mRNA levels (relative to Tub 9) in two lines overexpressing HDC1 under control of 35-S or Ubiquitin-10 promoters.
  • FIG. 7 Salkl 50126 and Sail1263E05 are not hdd knockouts.
  • A Position of T-DNA and primer pairs in the genomic DNA for Salk_150126 and Sail_1263_E05 lines.
  • B HDC1 mRNA levels in A. thaliana wildtype, Salk_150126 and Sail_1263_E05 using the primer pairs indicated in A. Rpll is RNA polymerasell (loading control). Asterisks indicate significant differences to the wild type (p ⁇ 0.05).
  • C Germination rates of A. thaliana wildtype (black), Salk_150126 (grey stripes) and Sail_1263_E05 (light grey stripes) on agar containing different concentrations of ABA. Bars are means +/- SE of at least 3 plates containing at least 50 seeds each. Note that neither of the lines shows ABA hypersensitivity.
  • FIG. 8 HDC1 de-sensitizes seedlings to salt, mannitol, ABA and PAC. Germination rates of A. thaliana wildtype (black), hdc1 -1 knockout (white) and HDC1 overexpressing (OX) lines (grey) on agar containing different concentrations of salt (NaCI, A), mannitol (B), ABA (C) or GA-biosynthesis inhibitor paclobutrazol (PAC, D). Germination rates in % reflect the number of seedlings that had developed cotyledons on day 6 after sowing, normalized to the total number of seeds sown. Bars are means ⁇ SE of at least 3 plates containing 50 seeds each. Asterisks indicate significant differences (p ⁇ 0.05) to wildtype. A photo of the seedlings is shown in Fig.9.
  • FIG. 9 A: Appearance of young A. thaliana seedlings on day 6 after sowing. Wildtype (upper third of plate), hdc1 -1 (centre) and OX (lower) seeds were imbibed and allowed to germinate on half strength Murashige Skoog medium without (control) or with 0.3 added. Pictures were taken on the same day as germination rate was scored. Note that without ABA, number and size of seedlings is similar for all lines.
  • B Transcript levels for embryogenesis related genes ABI3, FUS3 and LEC1 in wildtype (WT, black), hdd -1 knockout (KO, white) and HDC1 over-expressing (OX, grey) seedlings 2-6 days after germination (DAG). Bars represent means of 4 technical qPCR replicates with mRNA pooled from 50 seedlings. Asterisk indicates significant difference to wildtype (p ⁇ 0.05).
  • FIG. 10 HDA6 over-expression does not affect germination or growth.
  • A Germination rates of imbibed
  • B Transcript levels of HDA6 in wildtype and 35S::HDA6 lines.
  • C: Shoot weights (FW: fresh weight, DW: dry weight of 5- weeks old plants). Bars are means of 8 plants.
  • FIG. 1 Histone deacetylation is required for ABA-hyposensitivity. Germination rates of A. thaliana wildtype (B) and HDC1 overexpressing plants (B, C) on agar containing increasing concentrations of ABA with or without 0.3 or 3 ⁇ histone de-acetylation inhibitor trichostatin A (TSA). Other details as in Fig. 8.
  • FIG. 12 Knockout of HDC1 delays flowering without altering the plastochron.
  • A Plastochron of A. thaliana wildtype (black), hdd -1 knockout (white) and HDC1 OX plants (grey) growing on soil in long-day conditions. Bars are means of 3 plants ⁇ SE.
  • B Plant age at bolting. Bars are means ⁇ SE of 10-15 plants.
  • C Number of leaves at bolting. Bars are means ⁇ SE of 10-15 plants.
  • D FLC transcript levels on day 28. Bars are means ⁇ SE of 3 plants. Asterisks indicate differences to wildtype at p ⁇ 0.05.
  • FIG. 14 HDC1 enhances leaf surface of expanding rosette leaves in young plants.
  • Leaf surface areas of 2- weeks old A. thaliana wildtype (black), hdc1 -1 (white ) and HDC1 -OX (grey) plants grown on soil in long-day conditions. All plants had the same number of leaves (see Fig. 7A). Leaves were removed in order of appearance and analysed with Image J. Bars are means ⁇ SE of 3 plants. Asterisks indicate significant differences (p ⁇ 0.05) to wildtype.
  • FIG. 15 HDA6 knockdown affects plant growth without delaying leaf development.
  • A Fresh and dry weights of 4-weeks old
  • FIG. 16 HDC1 Knockout/Overexpression deregulates salt-responsive genes.Transcript levels of salt- responsive genes in A. thaliana wild type (WT; black), hdc1 -1 knockout (KO; white), and HDC1 overexpressing line (OX; gray). Plants were grown for 4 weeks in short-day conditions and subjected (+) or not (2) to 150 mM NaCI for 24 h in hydroponics. mRNA was pooled from three independently treated plant batches of five plants each. Each replicate treatment resulted in a significant increase of ABA (see Figure 17). Transcript levels were normalized to those of tubulin 9 (TUB9). Bars are means of four technical qPCR replicates 6 SE. Asterisks indicate significant differences to the wild type (P ⁇ 0.05). RAB18, RESPONSIVE TO ABA18.
  • FIG. 17 HDC1 has a small effect on ABA content after salt treatment.
  • A Shoot ABA content of wildtype (WT, black), hdc1 -1 knockout (KO, white) and HDC1 over-expressing (OX, grey). Plants were grown for 4 weeks in short day conditions and subjected (+) or not (-) to 150 mM NaCI for 24 h in hydroponics. Absolute results from three independently treated plant batches are shown.
  • B Relative change of ABA content in hdc1 -1 and HDC1 -overexpressing plants compared to wildtype. ABA content was normalized to the ABA content of salt-treated wildtype plants in the same batch.
  • FIG. 18 HDC1 determines H3K9/K14 acetylation status of ABA1 , DR4, PYL4 and RD29B. Relative amounts of DNA associated with acetylated H3K9/K14 for ABA1 , DR4, PYL4 and RD29B as determined by Ch lP-qPCR in A. thaliana wildtype (WT, black), hdd -1 knockout (KO, white) and HDC1 over-expressing (OX, grey) plants. Leaf tissue was pooled from 4-weeks old plants grown in 3 independent batches 12 plants each. Chromatin extracted and immunoprecipitated with anti-H3K9K14Ac.
  • qPCR-amplified ChlP-DNA was normalized to actin 2 and to input DNA (chromatin before immunoprecipitation). Bars are means of 4 technical qPCR-replicates ⁇ SE. Asterisks indicate significant differences to the wild type (p ⁇ 0.05).
  • FIG. 19 HDC1 increases plant growth in well-watered and in water-limited conditions.
  • A Rosette diameter and shoot weights (fresh weight; FW, dry weight: DW) of A. thaliana wildtype (black), hdd -1 knockout (white) and HDC1 OX plants (grey). Plants were grown on soil in short-day conditions. The water-limited regime consisted in reducing water supply from day 14 to achieve a continuous relative soil water content of -50% of the control condition until the end of the experiment at day 40. Bars are means ⁇ SE of at least 24 plants. Asterisks indicate differences to wildtype at p ⁇ 0.05.
  • Root and shoot weights of hydroponically grown plants growing in nutrient solution with 80 mM NaCI were 29 days (short-day conditions). The first time point is 6 hours after salt application. Control plants grown in parallel without salt are shown in Fig. 8. Bars are mean fresh weights (FW) ⁇ SE of 6 plants per line. Asterisks indicate differences to wildtype at p ⁇ 0.05. For determination of dry weight (DW) the tissues of 6 plants were pooled. Photos show plants of each line after 6 days in 80 mM NaCI.
  • FIG. 23 HDC1 increases yield under control conditions. Yield in gram per plant of wheat wildtype ("Control") and for 2 events (Event4 and Event5) performing better under control conditions.
  • FIG. 24 HDC1 has mRNA expression in transformed wheat plants. Event#1 and Event#2 clearly show mRNA expression. H stands for homozygous segregants, A stands for wild type segregants.
  • FIG. 25 HDC1 has mRNA expression in transformed wheat plants. Event#4 and Event#5 clearly show mRNA expression. H stands for homozygous segregants, A stands for wild type segregants. Detailed description
  • HDC1 HISTONE DEACETYLASE COMPLEX 1
  • HDC1 is a single copy gene from Arabidopsis thaliana that is conserved in single or low copy number in other plant species including important crops. It has partial homology to the yeast gene Rxt3, a confirmed but functionally uncharacterised member of the LRpd3 complex (Carrozza et al., 2005, Bba-Gene Struct Expr 1731 , 77-87; Chen et al., 2012, Curr Biol 22, 56-63).
  • HDC1 cannot be inferred from existing knowledge. Neither RXT3-type nor HDC1 - type genes have been functionally characterized to date, and neither of them contain any known functional motifs. Furthermore, the plant genes are considerably longer than the ancestral RXT3 genes and could have acquired new functions. The inventors have shown that HDC1 is ubiquitously expressed in all diploid tissues and localizes to the nucleus where it interacts with histone deactelylases HDA6 and HDA19. HDC1 was found to promote histone deacetylation as it appeared to be required for de-acetylation of lysine residues in histone 3.
  • HDC1 overexpression resulted in three basic phenotypes (i) ABA-insensitivity of post-germination growth in seedlings and of stress-induced ABA- synthesis in mature plants, (ii) enhanced vegetative growth (biomass production) both in well-watered and in water- limited soils, and (iii) accelerated flowering, while in hdd knockout mutants these features were oppositely affected. A yield increase could also be observed in wheat plants. This shows that the phenotypes were indeed caused by HDC1 , thereby identifying HDC1 as a critical determinant of plant growth, flowering and abiotic stress responses.
  • HDC1 histone deacetylation
  • stimulus e.g. ABA
  • activator e.g. transcription factor
  • Absence of HDC1 lowers the amount of stimulus required for de-repression but is not sufficient to activate transcription when stimulus and activator are absent (i.e. in control conditions).
  • HDC1 decreases the efficiency of a given amount of constitutive activator thereby reducing transcript levels.
  • HDC1 modulates ABA-sensitivity, growth and flowering by functioning as a universal scaffolding protein that enhances the apparent histone deacetylase activity by stabilizing the interaction of the enzymes with the substrate or with other regulatory proteins.
  • Hdd knockouts contrary to over-expression of an HDA19 homolog in rice, which increased growth but also produced a range of developmental abnormalities (Zhou et al. 2005, supra), no such abnormalities occurred in HDC1 -overexpressing plants. Hdd knockouts also did not reproduce aberrant developmental phenotypes observed in hda6/19 double mutants (Tian and Chen, 2001 , Proc. Natl. Acad. Aci.
  • the invention provides a method for increasing the tolerance of a plant, plant part, plant organ or plant cell to stress conditions, preferably mild or moderate stress conditions; or for reducing ABA sensitivity of a plant, plant part, plant organ or plant cell; or for increasing biomass or yield or growth rate of a plant, plant organ or plant part; or for accelerating flowering time of a plant; comprising the step of increasing the functional expression (i.e. the expression and/or activity) of HDC1 , i.e. a protein having the activity of the protein encoded by SEQ ID NO. 6, in said plant, plant part, plant organ or plant cell.
  • the functional expression i.e. the expression and/or activity
  • a protein having the activity of the protein with the amino acid sequence of SEQ ID NO. 6 relates to any functional HDC1 protein.
  • these include for example the plant HDC1 proteins as represented by by SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40 and SEQ ID NO. 41 ,
  • This also includes functional variants thereof, e.g.
  • proteins having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences cited above that encode a functional HDC1 protein.
  • Another example is based on the amino acid sequence enclosed by the nucleotide sequence of SEQ ID NO.: 42.
  • HDC1 proteins are ubiquitously expressed nuclear proteins of about 900 amino acids, of which the C-terminal half share sequence identity to the Rxt3-type proteins in green algae, protozoa and fungi (see figure 1 ), such as the 294- aa yeast protein Rxt3 (SEQ ID NO 4).
  • HDC1 has furthermore been shown to be required for histone de-ace tylation and to interact with various histone deacetylases (HDACs). Without intending to limit the invention to a particular mode of action, it is believed HDC1 functions as a relatively non-specific structural component to enhance the stability of histone deacetylation complexes, thereby increasing the efficiency of histone de-acetylation and downstream gene repression.
  • HDC1 is not required for basal HDAC activity, as knockouts are viable, but thought to titrate the efficiency of HDACs. Further, as an enhancer of HDAC activity HDC1 depends on the catalytic function of HDACs but decreases sensitivity of processes that involve HDAC function to histone deacetylase inhibitor compounds (e.g. TSA) and to hormones such as ABA.
  • TSA histone deacetylase inhibitor compounds
  • Increasing the expression and/or activity of an HDC1 protein can be achieved by modifying the endogenous gene or genes encoding such an HDC1 protein or by introducing a transgene, which when transcribed or expressed results in an increase of HDC1 protein expression and/or activity.
  • increasing the activity and or expression of HDC1 proteins in order to produce a plant or plant cell with increased tolerance to stress conditions or a plant with increased yield/biomass/growth or a plant with earlier flowering time can be achieved by providing that plant, or plant cell with a chimeric gene, which when expressed results in an increased activity and/or expression of a protein, e.g using the approaches as described above.
  • the invention provides a method for increasing the stress tolerance of a plant, plant part, plant organ or plant cell; or for increasing biomass or yield or growth of a plant, plant organ or plant part; or for accelerating flowering time of a plant, comprising the steps of expressing in said plant, plant part, plant organ or plant cell a chimeric gene comprising the following operably linked elements:
  • a nucleic acid which when transcribed results in an increased activity and/or expression of a protein having the activity of the protein encoded by SEQ ID NO. 6;
  • a nucleic acid which when transcribed results in an increased activity and/or expression of a protein having the activity of the protein encoded by SEQ ID NO. 6 can encode an activating transcription factor that targets the promoter of the endogenous HDC1 gene present in the plant (e.g. the promoter such as represented by SEQ ID NO. 1 ), such that expression of the endogenous HDC1 gene is increased.
  • Such transcription factors can be designed for example by coupling a non-specific transcription enhancer to a sequence specific DNA binding protein.
  • Such techniques for designing transcription factors with a particular desired site specificity are e.g. described in Bogdanova and Voytas (201 1 , Science 333, p1843-1846) and references therein.
  • the nucleic acid can itself encode a H DC1 protein, thereby increasing the amount of functional HDC1 protein in the cell, such as proteins comprising the amino acid sequence of SEQ ID NO. 6, or functional variants thereof, e.g. proteins having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences cited above.
  • proteins comprising the amino acid sequence of SEQ ID NO. 6, or functional variants thereof, e.g. proteins having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences cited above.
  • the nucleic acid encodes an HDC1 protein and comprises the nucleotide sequence of SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 1 1 , SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21 , SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 , SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37 and SEQ ID NO. 39, or variants thereof, e.g.
  • nucleotide sequences having at least 50%, at least 60% , at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the nucleotide sequences cited above and which encode a functional HDC1 protein.
  • sequence identity of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (x100) divided by the number of positions compared.
  • a gap i.e., a position in an alignment where a residue is present in one sequence but not in the other, is regarded as a position with non-identical residues.
  • the "optimal alignment” of two sequences is found by aligning the two sequences over the entire length according to the Needleman and Wunsch global alignment algorithm (Needleman and Wunsch, 1970, J Mol Biol 48(3):443-53) in The European Molecular Biology Open Software Suite (EMBOSS, Rice et al.
  • HDC1 genes encoding HDC1 other than the genes encoding protein with amino acid sequences or having the coding sequences mentioned above.
  • homologous nucleotide sequence may be identified and isolated by hybridization under stringent conditions using as probes identified nucleotide sequences.
  • High stringency conditions can be provided, for example, by hybridization at 65°C in an aqueous solution containing 6x SSC (20x SSC contains 3.0 M NaCI, 0.3 M Na-citrate, pH 7.0), 5x Denhardt's (100X Denhardt's contains 2% Ficoll, 2% Polyvinyl pyrollidone, 2% Bovine Serum Albumin), 0.5% sodium dodecyl sulphate (SDS), and 20 ⁇ g/ml denaturated carrier DNA (single-stranded fish sperm DNA, with an average length of 120 - 3000 nucleotides) as non- specific competitor. Following hybridization, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridization temperature in 0.2-0.1 ⁇ SSC, 0.1 % SDS.
  • Low stringency refers to conditions equivalent to hybridization in the above described solution at about 50- 52°C. Low stringency washing may be done at the hybridization temperature in 2x SSC, 0.1 % SDS. See also Sambrook et al. (1989) and Sambrook and Russell (2001).
  • chimeric gene refers to a gene that is made up of heterologous elements that are operably linked to enable expression of the gene, whereby that combination is not normally found in nature.
  • heterologous refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources.
  • a promoter is heterologous with respect to an operably linked nucleic acid sequence, such as a coding sequence, if such a combination is not normally found in nature.
  • a particular sequence may be "heterologous" with respect to a cell or organism into which it is inserted (i.e. does not naturally occur in that particular cell or organism).
  • operably linked means that said elements of the chimeric gene are linked to one another in such a way that their function is coordinated and allows expression of the coding sequence, i.e. they are functionally linked.
  • a promoter is functionally linked to another nucleotide sequence when it is capable of ensuring transcription and ultimately expression of said other nucleotide sequence.
  • Two proteins encoding nucleotide sequences e.g. a transit peptide encoding nucleic acid sequence and a nucleic acid sequence encoding a protein having HDC1 activity, are functionally or operably linked to each other if they are connected in such a way that a fusion protein of first and second protein or polypeptide can be formed.
  • a gene e.g. the chimeric gene of the invention, is said to be expressed when it leads to the formation of an expression product.
  • An expression product denotes an intermediate or end product arising from the transcription and optionally translation of the nucleic acid, DNA or RNA, coding for such product, e. g. the second nucleic acid described herein.
  • a DNA sequence under control of regulatory regions, particularly the promoter is transcribed into an RNA molecule.
  • An RNA molecule may either itself form an expression product or be an intermediate product when it is capable of being translated into a peptide or protein.
  • a gene is said to encode an RNA molecule as expression product when the RNA as the end product of the expression of the gene is, e.
  • RNA expression products include inhibitory RNA such as e. g. sense RNA (co-suppression), antisense RNA, ribozymes, miRNA or siRNA, mRNA, rRNA and tRNA.
  • a gene is said to encode a protein as expression product when the end product of the expression of the gene is a protein or peptide.
  • promoters may be used to promote the transcription of the nucleic acid of the invention, i.e. the nucleic acid which when transcribed results in an increased activity and/or expression of an HDC1 protein.
  • Such promoters include for example constitutive promoters, inducible promoters (e.g. stress-inducible promoters, drought-inducible promoters, hormone-inducible promoters, chemical-inducible promoters, etc.), tissue-specific promoters, developmental ⁇ regulated promoters and the like.
  • a plant expressible promoter can be a constitutive promoter, i.e. a promoter capable of directing high levels of expression in most cell types (in a spatio-temporal independent manner).
  • plant expressible constitutive promoters include promoters of bacterial origin, such as the octopine synthase (OCS) and nopaline synthase (NOS) promoters from Agrobacterium, but also promoters of viral origin, such as that of the cauliflower mosaic virus (CaMV) 35S transcript (Hapster et al., 1988, Mol. Gen. Genet. 212: 182-190) or 19S RNAs genes (Odell et al., 1985, Nature.
  • CCS octopine synthase
  • NOS nopaline synthase
  • promoters of plant origin mention will be made of the promoters of the plant ribulose-biscarboxylase/oxygenase (Rubisco) small subunit promoter (US 4,962,028; W099/25842) from zea mays and sunflower, the promoter of the Arabidopsis thaliana histone H4 gene (Chaboute et al., 1987), the ubiquitin promoters (Holtorf et al., 1995, Plant Mol. Biol.
  • Rubisco ribulose-biscarboxylase/oxygenase
  • a variety of plant gene promoters that regulate gene expression in response to environmental, hormonal, chemical, developmental signals, and in a tissue-active manner can be used for expression of a sequence in plants.
  • Choice of a promoter is based largely on the phenotype of interest and is determined by such factors as tissue (e.g., seed, fruit, root, pollen, vascular tissue, flower, carpel, etc.), inducibility (e.g., in response to wounding, heat, cold, drought, light, pathogens, etc.), timing, developmental stage, and the like.
  • Additional promoters that can be used to practice this invention are those that elicit expression in response to stresses, such as the RD29 promoters that are activated in response to drought, low temperature, salt stress, or exposure to ABA (Yamaguchi-Shinozaki et al., 2004, Plant Cell, Vol. 6, 251 -264; WO12/101 1 18), but also promoters that are induced in response to heat (e.g., see Ainley et al. (1993) Plant Mol. Biol. 22: 13-23), light (e.g., the pea rbcS-3A promoter, Kuhlemeier et al.
  • stresses such as the RD29 promoters that are activated in response to drought, low temperature, salt stress, or exposure to ABA (Yamaguchi-Shinozaki et al., 2004, Plant Cell, Vol. 6, 251 -264; WO12/101 1 18), but also promoters that are induced in response to heat (e.g., see
  • timing of the expression can be controlled by using promoters such as those acting at senescence (e.g., see Gan and Amasino (1995) Science 270: 1986-1988); or late seed development (e.g., see Odell et al. (1994) Plant Physiol. 106: 447458).
  • promoters such as those acting at senescence (e.g., see Gan and Amasino (1995) Science 270: 1986-1988); or late seed development (e.g., see Odell et al. (1994) Plant Physiol. 106: 447458).
  • salt-inducible promoters such as the salt-inducible NHX1 promoter of rice landrace Pokkali (PKN) (Jahan et al., 6 th International Rice Genetics symposium, 2009, poster abstract P4-37), the salt inducible promoter of the vacuolar ⁇ -pyrophosphatase from Thellungiella halophila (TsVP1 ) (Sun et al., BMC Plant Biology 2010, 10:90), the salt-inducible promoter of the Citrus sinensis gene encoding phospholipid hydroperoxide isoform gpxl (Avsian-Kretchmer et al., Plant Physiology July 2004 vol. 135, p1685-1696).
  • PPN salt-inducible NHX1 promoter of rice landrace Pokkali
  • TsVP1 Thellungiella halophila
  • TsVP1 Thellungiella halophila
  • tissue-specific and/or developmental stage-specific promoters are used, e.g., promoter that can promote transcription only within a certain time frame of developmental stage within that tissue. See, e.g., Blazquez (1998) Plant Cell 10:791 -800, characterizing the Arabidopsis LEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77 , describing the transcription factor SPL3, which recognizes a conserved sequence motif in the promoter region of the A. thaliana floral meristem identity gene API; and Mandel (1995) Plant Molecular Biology, Vol. 29, pp 995-1004, describing the meristem promoter elF4.
  • Tissue specific promoters which are active throughout the life cycle of a particular tissue can be used.
  • the nucleic acids of the invention are operably linked to a promoter active primarily only in cotton fiber cells
  • the nucleic acids of the invention are operably linked to a promoter active primarily during the stages of cotton fiber cell elongation, e.g., as described by Rinehart (1996) supra.
  • the nucleic acids can be operably linked to the Fbl2A gene promoter to be preferentially expressed in cotton fiber cells (Ibid) . See also, John (1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Patent Nos.
  • Root- specific promoters may also be used to express the nucleic acids of the invention.
  • Examples of root-specific promoters include the promoter from the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol. 123:39-60) and promoters such as those disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186.
  • a leaf-specific promoter see, e.g., Busk (1997) Plant J. 1 1 :1285 1295, describing a leaf-specific promoter in maize
  • the ORF 13 promoter from Agrobacterium rhizogenes which exhibits high activity in roots,
  • a tomato promoter active during fruit ripening, senescence and abscission of leaves a guard-cell preferential promoter e.g. as described in PCT/EP12/065608, and, to a lesser extent, of flowers can be used (see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specific promoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol. Biol.
  • the Blec4 gene from pea which is active in epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa making it a useful tool to target the expression of foreign genes to the epidermal layer of actively growing shoots or fibers
  • the ovule-specific BELI gene see, e.g., Reiser (1995) Cell 83:735-742, GenBank No. U39944)
  • the promoter in Klee, U.S. Patent No. 5,589,583, describing a plant promoter region is capable of conferring high levels of transcription in meristematic tissue and/or rapidly dividing cells.
  • tissue specific promoters that may be used according to the invention include: seed-specific promoters (such as the napin, phaseolin or DC3 promoter described in U .S. Pat. No. 5,773,697), fruit-specific promoters that are active during fruit ripening (such as the dru 1 promoter (U.S. Pat. No. 5,783,393), or the 2AI 1 promoter (e.g., see U.S. Pat. No. 4,943,674) and the tomato polygalacturonase promoter (e.g., see Bird et al. (1988) Plant Mol. Biol. 1 1 : 651 -662), flower- specific promoters (e.g., see Kaiser et al. (1995) Plant Mol. Biol.
  • seed-specific promoters such as the napin, phaseolin or DC3 promoter described in U .S. Pat. No. 5,773,697
  • fruit-specific promoters that are active during fruit ripening such as the dru 1 promoter (
  • pollen-active promoters such as PTA29, PTA26 and PTAI 3 (e.g., see U.S. Pat. No. 5,792,929) and as described in e.g. Baerson et al. (1994 Plant Mol. Biol. 26: 1947-1959), promoters active in vascular tissue (e.g., see Ringli and Keller (1998) Plant Mol. Biol. 37: 977-988), carpels (e.g., see Ohl et al. (1990) Plant Cell 2:), pollen and ovules (e.g., see Baerson et al. (1993) Plant Mol. Biol. 22: 255- 267).
  • PTA29 e.g., see U.S. Pat. No. 5,792,929
  • promoters active in vascular tissue e.g., see Ringli and Keller (1998) Plant Mol. Biol. 37: 977-988
  • carpels e.g., see Ohl et al. (1990) Plant
  • plant promoters which are inducible upon exposure to plant hormones, such as auxins, are used to express the nucleic acids used to practice the invention.
  • the invention can use the auxin- response elements El promoter fragment (AuxREs) in the soybean ⁇ Glycine max L.) (Liu (1997) Plant Physiol. 1 15:397- 407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Streit (1997) Mol. Plant Microbe Interact.
  • ABA abscisic acid
  • Further hormone inducible promoters include auxin-inducible promoters (such as that described in van der Kop et al. (1999) Plant Mol. Biol. 39: 979-990 or Baumann et al., (1999) Plant Cell 1 1 : 323-334), cytokinin-inducible promoter (e.g., see Guevara-Garcia (1998) Plant Mol. Biol. 38: 743-753), promoters responsive to gibberellin (e.g., see Shi et al. (1998) Plant Mol. Biol. 38: 1053-1060, Willmott et al. (1998) Plant Molec. Biol. 38: 817-825) and the like.
  • gibberellin e.g., see Shi et al. (1998) Plant Mol. Biol. 38: 1053-1060, Willmott et al. (1998) Plant Molec. Biol. 38: 817-825
  • nucleic acids used to practice the invention can also be operably linked to plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics.
  • plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics.
  • the maize ln2-2 promoter activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem.
  • Coding sequence can be under the control of, e.g., a tetracycline-inducible promoter, e.g.
  • transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 1 1 :465 73); or, a salicylic acid-responsive element (Stange (1997) Plant J. 1 1 :1315-1324).
  • arginine decarboxylase gene Masgrau (1997) Plant J. 1 1 :465 73); or, a salicylic acid-responsive element (Stange (1997) Plant J. 1 1 :1315-1324).
  • chemically- ⁇ e.g. , hormone- or pesticide- ) induced promoters i.e., promoter responsive to a chemical which can be applied to the transgenic plant in the field
  • expression of a polypeptide of the invention can be induced at a particular stage of development of the plant.
  • Use may also be made of the estrogen-inducible expression system as described in US patent 6,784,340 and Zuo et al. (2000, Plant
  • the a promoter may be used whose host range is limited to target plant species, such as corn, rice, barley, wheat, potato or other crops, inducible at any stage of development of the crop.
  • a tissue-specific plant promoter may drive expression of operably linked sequences in tissues other than the target tissue.
  • a tissue-specific promoter that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well, is used.
  • promoter elements as e.g. described on http://arabidopsis.med.ohio-state.edu/AtcisDB/bindingsites.html., which in combination should result in a functional promoter.
  • IME intron-mediated enhancement
  • introns have also been described to affect translation, to function as promoters or as position and orientation independent transcriptional enhancers (Chaubet-Gigot et al., 2001 , Plant Mol Biol. 45(1 ):17-30, p27-28).
  • genes containing such introns include the 5' introns from the rice actin 1 gene (see US5641876), the rice actin 2 gene, the maize sucrose synthase gene (Clancy and Hannah, 2002, Plant Physiol. 130(2):918-29), the maize alcohol dehydrogenase-1 (Adh-1 ) and Bronze-1 genes (Callis et al. 1987 Genes Dev. 1 (10):1 183-200; Mascarenhas et al. 1990, Plant Mol Biol.
  • Suitable regulatory sequences include 5' UTRs.
  • a 5'UTR also referred to as leader sequence, is a particular region of a messenger RNA (mRNA) located between the transcription start site and the start codon of the coding region. It is involved in mRNA stability and translation efficiency.
  • mRNA messenger RNA
  • the 5' untranslated leader of a petunia chlorophyll a/b binding protein gene downstream of the 35S transcription start site can be utilized to augment steady-state levels of reporter gene expression (Harpster et al., 1988, Mol Gen Genet. 212(1 ):182-90).
  • WO95/006742 describes the use of 5' non-translated leader sequences derived from genes coding for heat shock proteins to increase transgene expression.
  • the chimeric gene may also comprise a 3' end region, i.e. a transcription termination or polyadenylation sequence, operable in plant cells.
  • a transcription termination or polyadenylation sequence use may be made of any corresponding sequence of bacterial origin, such as for example the nos terminator of Agrobacterium tumefaciens, of viral origin, such as for example the CaMV 35S terminator, or of plant origin, such as for example a histone terminator as described in published Patent Application EP 0 633 317 A1.
  • the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
  • the 3' end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
  • Other measures to increase the expression that may be applied is optimizing the coding region for expression in the target organism, which may include adapting the codon usage , CG content, and elimination of unwanted nucleotide sequences (e.g. premature polyadenylation signals , cryptic intron splice sites, ATTTA pentamers, CCAAT box sequences, sequences that effect pre-mRNA splicing by secondary RNA structure formation such as long CG or AT stretches).
  • unwanted nucleotide sequences e.g. premature polyadenylation signals , cryptic intron splice sites, ATTTA pentamers, CCAAT box sequences, sequences that effect pre-mRNA splicing by secondary RNA structure formation such as long CG or AT stretches.
  • the coding sequence of the chimeric gene may further be modified as to increase protein stability, prevent protein degradation, enhance protein activity of the encoded HDC1 protein, for instance by introducing or deleting sites involved in post-translational modifications, such as sumoylation, ubiquitination, phosphorylation etc.
  • the HDC1 sequence as represented by SEQ ID NO. 6 contains a relatively high number of predicted sumoylation sites, suggesting that sumoylation plays an important role in maintaining HDC1 protein levels/activity. About 20% of lysines are concerned, compared to 7-14% in a random selection of proteins of similar length. The probability scores are extremely high (e.g. 94% for K273,K426, K192) and the sites are well conserved in HDC1 sequences of other plant species such as the HDC1 sequences described above .
  • the nucleic acid of the chimeric gene encoding the HDC1 protein can be modified such that the encoded HDC1 protein interacts more tightly to HDAC proteins, for example by optimizing HDAC binding sites or introducing more HDAC binding sites.
  • increasing the functional expression (i.e. the expression and/or activity) of HDC1 i.e. a protein having the activity of the protein encoded by SEQ ID NO. 6, can be achieved by modifying the endogenous gene(s) encoding an HDC1 protein. This can be done through, for example, T-DNA activation tagging, mutagenesis (e.g. EMS mutagenesis) or by targeted genome engineering technologies.
  • the endogenous promoter can be modified such that it drives higher levels of expression, or the endogenous promoter can be replaced with a stronger promoter, or mutations can be introduced into the coding region that enhance mRNA stability, translation efficiency, protein activity and/or stability, similar to the above described methods for enhancing the expression of the introduced chimeric gene.
  • T-DNA activation tagging is a method to activate endogenous genes by random insertion of a T-DNA carrying promoter or enhancer elements, which can cause transcriptional activation of flanking plant genes.
  • the method can consist of generating a large number of transformed plants or plant cells using a specialized T-DNA construct, followed by selection for the desired phenotype.
  • Targeted genome engineering refers to generating intended and directed modifications into the genome. Such intended modifications can be insertions at specific genomic locations, deletions of specific endogenous sequences, and replacements of endogenous sequences. Targeted genome engineering can be based on homologous recombination. Targeted genome engineering to increase the functional expression of the HDC1 endogene can consist of insertion of a promoter, stronger than the endogenous promoter, in front of the HDC1 coding sequence, or insert an enhancer to increase promoter activity. Such techniques can also be applied e.g. to insert elements increasing RNA stability or enhancing translation of the encoded mRNA, or modify the coding sequence to enhance translation, protein stability and activity, similar to the above described methods for enhancing the expression of the introduced chimeric gene.
  • mutagenesis refers to the process in which plant cells are subjected to a technique which induces mutations in the DNA of the cells, such as contact with a mutagenic agent, such as a chemical substance (such as ethyl methylsulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation (neutrons (such as in fast neutron mutagenesis, etc.), alpha rays, gamma rays (such as that supplied by a Cobalt 60 source), X-rays, UV-radiation, etc.), or targeted mutagenesis methods e.g. via oligonucleotides (e.g. KeyBase® technology). These methods can also be applied to modufy the endogenous HDC1 encoding gene(s) as desired.
  • a mutagenic agent such as a chemical substance (such as ethyl methylsulfonate (EMS), ethylnitrosoure
  • transcript e.g. an mRNA
  • expression of a transcript can be measured according to various methods known in the art such as (quantitative) RT-PCR, northern blotting, microarray analysis, western blotting, ELISA and the like.
  • Increased expression refers to increase in expression level of at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 40%, or at least 50% or even more. Said increase is an increase with respect to the expression in control plants.
  • Stress conditions refers e.g.
  • abiotic stress e.g., drought, waterlogging, submergence, high light conditions, high UV radiation, increased hydrogen peroxide levels, extreme (high or low) temperatures, ozone and other atmospheric pollutants, soil salinity or heavy metals, hypoxia, anoxia, osmotic stress, oxidative stress, low nutrient levels such as nitrogen or phosphor etc.
  • biotic stress e.g., pathogen or pest infection including infection by fungi, viruses, bacteria, insects, nematodes, mycoplasms and mycoplasma like organisms, etc.
  • Stress may also be imposed by hormones such as ABA or compound influencing hormone activity.
  • Drought, salinity, extreme temperatures, high light stress and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms.
  • Rabbani et al. Plant Physiol (2003) 133: 1755- 1767
  • Drought, salinity, extreme temperatures, high light stress and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms.
  • Rabbani et al. Plant Physiol (2003) 133: 1755- 1767
  • Oxidative stress which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturing of functional and structural proteins.
  • these diverse environmental stresses often activate similar cell signalling pathways and cellular responses, such as the production of stress proteins, up- regulation of anti-oxidants, accumulation of compatible solutes and growth arrest.
  • an increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various mild or moderate stress conditions compared to control plants. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress or chronic stress, the plant may even stop growing altogether.
  • the condition of moderate stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth.
  • Moderate stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 1 1 % or 10% or less when compared to the control plant under non-stress conditions.
  • Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants.
  • the compromised growth induced by moderate stress is often an undesirable feature for agriculture, moderate stresses are the biotic and/or abiotic (environmental) stresses to which a plant is exposed under standard agricultural conditions.
  • the stress as described in the Examples below are considered to constitute moderate or moderate stress conditions.
  • the term "non-stress" conditions as used herein are those environmental conditions that allow optimal growth of plants.
  • the effects on the plant of moderate stress can be compensated for by reducing the ABA sensitivity of a plant, as is the case when the activity and/or expression of the HDC1 protein is increased according to the present invention.
  • severe stress cannot be compensated for by reducing ABA sensitivity, and in such cases it may be preferred to decrease the activity and or expression of the HDC1 protein of the invention, as will be set forth further below.
  • a "control plant” as used herein is generally a plant of the same species which has wild-type levels of HDC1 .
  • Wild-type levels of HDC1 refers to the typical levels of HDC1 protein in a plant as it most commonly occurs in nature. Said control plant has thus not been provided either with a nucleic acid molecule which when expressed increases the expression and/or activity of HDC1 , nor has it been provided with a nucleic acid molecule which when expressed decreases the expression and/or activity of HDC1 .
  • Yield or biomass refers to seed number/weight, fruit number/weight, fresh weight, dry weight, leaf number/area, plant height, branching, boll number/size, fiber length, seed oil content, seed protein content, seed carbohydrate content.
  • An increased growth rate as used herein refers to a period of increased growth or allocation to one or more of these cells or tissues that comprise the aforementioned plant organs.
  • An increase in biomass or yield or growth can be an increase of at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 40% , or at least 50%. Said increase is an increase with respect to biomass or yield or growth of control plants.
  • Abscisic acid is a phytohormone which functions in many plant developmental processes, including seed dormancy. Furthermore, ABA mediates stress responses in plants in reaction to water stress, high-salt stress, cold stress (Mansfield 1987, p. 41 1-430. In: P.J. Davies (ed.). Plant hormones and their role in plant growth and development. Martinus Nijhoff Publishers, Dordrecht; Yamaguchi-Shinozaki 1993, Plant Physiol. 101 , 1 1 19-1 120; Yamaguchi- Shinozaki 1994, Plant Cell 6, 251 -264) and plant pathogens (Seo and Koshiba, 2002, Trends Plant Sci. 7, 41 ⁇ 8).
  • ABA is a sesquiterpenoid (15-carbon) which is partially produced via the mevalonic pathway in chloroplasts and other plastids. It is synthesized partially in the chloroplasts and accordingly, biosynthesis primarily occurs in the leaves. The production of ABA is increased by stresses such as water loss and freezing temperatures. It is believed that biosynthesis occurs indirectly through the production of carotenoids.
  • Physiological responses known to be associated with abscisic acid include stimulation of the closure of stomata, inhibition of seedling or shoot growth, induction of storage protein synthesis in seeds and inhibition of the effect of gibberellins on stimulating de novo synthesis of a-amylase.
  • Basic ABA levels may differ considerably from plant to plant.
  • the basal concentration of ABA in non-stressed Arabidopsis leaves is 2 to 3 ng/g fresh weight (Lopez-Carbonell and Jauregui, 2005). Under water-stress conditions, the ABA concentration reaches 10 to 21 ng/g fresh weight.
  • ABA sensitivity can be measured e.g. as described herein below. ABA sensitivity can also be measured by measurement of stomatal aperture (Zhang et al. 2009, EurAsia J BioSci 3, 10-16), measurement of ion current s (Armstrong et al 1995, PNAS 92:95204; Marten et al. 2007, Plant Physiol. Vol. 143, 28037) or measurement of ABA- dependent gene expression by microarrays, RNA-sequencing, RT-PCR or RNA gel blotting (Hoth et al. 2002, Journal of Cell Science 1 15, 48914900).
  • Decrease in ABA sensitivity can be a decrease of at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20% , or at least 25%, or at least 30%, or at least 40%, or at least 50%. Said decrease is a decrease with respect to ABA sensitivity of control plants.
  • the invention provides methods for enhancing survival of a plant, plant part, plant organ or plant cell under severe stress conditions, methods for enhancing recovery after severe stress of a plant, plant part, plant organ or plant cell , or methods for delaying the flowering time of a plant, comprising the step of decreasing the functional expression (expression and/or activity) protein having the activity of the protein encoded by SEQ ID NO. 6 (an HDC1 protein) in the plant, plant part, plant organ or plant cell.
  • HDC1 downregulation e.g. knockout
  • HDC1 downregulation increases ABA sensitivity
  • HDC1 downregulation under severe stress by increasing ABA sensitivity
  • HDC1 downregulation is inducible, as plants with constitutive low levels of HDC1 and concomitant ABA hypersensitivity are thought to have a growth penalty under control conditions.
  • Reduce or eliminate the activity of HDC1 in a plant or plant cell can e,g be achieved by introducing a nucleic acid into the plant or plant cell that may inhibit the expression or function of the HDC1 polypeptide directly, by preventing transcription or translation of an HDC1 messenger RNA, or indirectly, by encoding a polypeptide that inhibits the transcription or translation of an HDC1 gene encoding a HDC1 polypeptide.
  • Such nucleic acids are said to encode HDC1 -inhibitory RNA molecules.
  • Methods for inhibiting or eliminating the expression of a gene in a plant are well known in the art, and any such method may be used in the present invention to inhibit the expression of the HDC1 polypeptide.
  • a nucleic acid that encodes a polypeptide that inhibits the activity of an HDC1 polypeptide is introduced into a plant or plant cell. Many methods may be used to reduce or eliminate the activity of a HDC1 polypeptide.
  • the expression of HDC1 is inhibited if the transcript or protein level is statistically lower than the transcript or protein level of HDC1 in a plant that has not been modified to inhibit the expression of that HDC1 .
  • the transcript or protein level of the HCD1 may be less than 50%, less than 40% , less than 30%, less than 20%, less than 10%, or less than 5% of the mRNA or protein level of the same HDC1 in a plant that is not a mutant or that has not been modified to inhibit the expression of that HDC1 .
  • a nucleic acid is introduced into a plant or plant cell that upon induction of expression, inhibits the expression of HDC1 in the plant or plant cell.
  • Examples of nucleic acids that inhibit the expression of an HDC1 polypeptide are given below.
  • inhibition of the expression of an HDC1 polypeptide may be obtained by sense suppression or cosuppression.
  • a chimeric gene or expression cassette is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding an HDC1 polypeptide in the "sense" orientation.
  • the nucleic acid used for cosuppression may correspond to all or part of the sequence encoding the HDC1 polypeptide, all or part of the 5' and/or 3' untranslated region of an HDC1 polypeptide transcript or all or part of both the coding sequence and the untranslated regions of a transcript encoding an HDC1 polypeptide.
  • a nucleic acid used for cosuppression or other gene silencing methods may share 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, 90%, 89%, 88%, 87%, 85%, 80%, or less sequence identity with the target sequence.
  • portions of the nucleic acids e.g.,SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 1 1 , SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21 , SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 , SEQ ID NO. 33, SEQ ID NO.
  • 35, SEQ ID NO. 37 or SEQ ID NO. 39 are used to disrupt the expression of the target gene, generally, sequences of at least 15, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, or 1000 contiguous nucleotides or greater may be used.
  • the chimeric gene is designed to eliminate the start codon of the polynucleotide so that no protein product will be translated. Multiple plant lines transformed with the cosuppression chimeric gene can then be screened to identify those that show the desired (inducible) inhibition of HDC1 polypeptide expression.
  • inhibition of the expression of the HDC1 polypeptide may be obtained by antisense suppression.
  • the chimeric gene or expression cassette is designed to express an RNA molecule complementary to all or part of a messenger RNA encoding the HDC1 polypeptide. Overexpression of the antisense RNA molecule can result in reduced expression of the native gene.
  • the polynucleotide for use in antisense suppression may correspond to all or part of the complement of the sequence encoding the HDC1 polypeptide, all or part of the complement of the 5' and/or 3' untranslated region of the HDC1 transcript or all or part of the complement of both the coding sequence and the untranslated regions of a transcript encoding the HDC1 polypeptide.
  • the antisense nucleic acid may be fully complementary (i.e. 100% identical to the complement of the target sequence) or partially complementary (i.e.
  • SEQ ID NO. 5 SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 1 1 , SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21 , SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 , SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37 or SEQ ID NO.
  • portions of the antisense nucleotides may be used to disrupt the expression of the target gene.
  • sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, 300, 400, 450, 500, 550 or greater may be used.
  • Multiple plant lines transformed with the antisense chimeric gene can then be screened to identify those that show the desired (inducible) inhibition of HDC1 polypeptide expression.
  • inhibition of the expression of an HDC1 polypeptide may be obtained by double-stranded RNA (dsRNA) interference.
  • dsRNA interference a sense RNA molecule like that described above for cosuppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA.
  • Expression of the sense and antisense molecules can be accomplished by designing the chimeric gene to comprise both a sense sequence and an antisense sequence. Alternatively, separate chimeric genes may be used for the sense and antisense sequences.
  • dsRNA interference chimeric gene or chimeric genes are then screened to identify plant lines that show the desired (inducible) inhibition of HDC1 polypeptide expression.
  • Methods for using dsRNA interference to inhibit the expression of endogenous plant genes are described in WO9949029, WO9953050, W09961631 and WO0049035, each of which is herein incorporated by reference.
  • inhibition of the expression of an HDC1 polypeptide may be obtained by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference.
  • hpRNA hairpin RNA
  • ihpRNA intron-containing hairpin RNA
  • the chimeric gene is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single-stranded loop region and a base-paired stem.
  • the base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene whose expression is to be inhibited, and an antisense sequence that is fully or partially complementary to the sense sequence.
  • the antisense sequence may be located "upstream" of the sense sequence (i.e. the antisense sequence may be closer to the promoter driving expression of the hairpin RNA than the sense sequence).
  • the base-paired stem region may correspond to a portion of a promoter sequence controlling expression of the gene to be inhibited.
  • a nucleic acid designed to express an RNA molecule having a hairpin structure comprises a first nucleotide sequence and a second nucleotide sequence that is the complement of the first nucleotide sequence, and wherein the second nucleotide sequence is in an inverted orientation relative to the first nucleotide sequence.
  • the base-paired stem region of the molecule generally determines the specificity of the RNA interference.
  • the sense sequence and the antisense sequence are generally of similar lengths but may differ in length.
  • sequences may be portions or fragments of at least 10, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 70, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 500, 600, 700, 800, 900 nucleotides in length, or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb in length.
  • the loop region of the chimeric gene may vary in length.
  • the loop region may be at least 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900 nucleotides in length, or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb in length.
  • hpRNA molecules are highly efficient at inhibiting the expression of endogenous genes and the RNA interference they induce is inherited by subsequent generations of plants. See, for example, Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38. A transient assay for the efficiency of hpRNA constructs to silence gene expression in vivo has been described by Panslita, et al. (2003) Mol. Biol. Rep. 30: 135-140, herein incorporated by reference.
  • the interfering molecules have the same general structure as for hpRNA, but the RNA molecule additionally comprises an intron in the loop of the hairpin that is capable of being spliced in the cell in which the ihpRNA is expressed.
  • the use of an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference. See, for example, Smith ef al (2000) Nature 407:319-320. In fact, Smith ef a/, show 100% suppression of endogenous gene expression using ihpRNA-mediated interference.
  • the intron is the ADHI intron 1.
  • the chimeric gene for hpRNA interference may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA.
  • the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene.
  • it is the loop region that determines the specificity of the RNA interference. See, for example, WO0200904 herein incorporated by reference.
  • Amplicon chimeric genes comprise a plant virus-derived sequence that contains all or part of the target gene but generally not all of the genes of the native virus.
  • the viral sequences present in the transcription product of the chimeric gene allow the transcription product to direct its own replication.
  • the transcripts produced by the amplicon may be either sense or antisense relative to the target sequence (i.e., the messenger RNA for the HDC1 polypeptide). Methods of using amplicons to inhibit the expression of endogenous plant genes are described, for example, in US6635805, which is herein incorporated by reference.
  • the nucleic acid expressed by the chimeric gene of the invention is catalytic RNA or has ribozyme activity specific for the messenger RNA of theHDCI polypeptide.
  • the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in reduced expression of the HDC1 polypeptide. This method is described, for example, in US4987071 , herein incorporated by reference.
  • inhibition of the expression of a HDC1 polypeptide may be obtained by RNA interference by expression of a nucleic acid encoding a micro RNA (miRNA).
  • miRNAs are regulatory agents consisting of about 22 ribonucleotides. miRNA are highly efficient at inhibiting the expression of endogenous genes. See, for example Javier ef a/ (2003) Wafure 425 :257-263, herein incorporated by reference.
  • the chimeric gene is designed to express an RNA molecule that is modeled on an endogenous pre-miRNA gene wherein the endogenous miRNA and miRNA* sequence are replaced by sequences targeting the HDC1 mRNA.
  • the miRNA gene encodes an RNA that forms a hairpin structure containing a 18-22-nucleotide, e.g. 21 nucleotide, sequence that is complementary to another endogenous gene (target sequence).
  • target sequence e.g. 21 nucleotide
  • the 18-22-nucleotide sequence is selected from the target transcript sequence and contains 18-22 nucleotides of said target sequence in sense orientation (the miRNA* sequence) and a corresponding antisense sequence that is complementary to the sense sequence and complementary to the target mRNA (the miRNA sequence).
  • the miRNA sequence No perfect complementarity between the miRNA and its target is required, but some mismatches are allowed. Up to 4 mismatches between the miRNA and miRNA* sequence are also allowed.
  • miRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants.
  • the nucleic acid encodes a zinc finger protein that binds to a gene encoding an HDC1 polypeptide, resulting in reduced expression of the gene.
  • the zinc finger protein binds to a regulatory region of an HDC1 gene.
  • the zinc finger protein binds to a messenger RNA encoding an HDC1 polypeptide and prevents its translation.
  • the TALE protein binds to a regulatory region of an HDC1 gene.
  • the TALE protein binds to a messenger RNA encoding an HDC1 polypeptide and prevents its translation.
  • polypeptides or nucleic acids encoding polypeptides can be introduced into a plant, wherein the encoded polypeptide is capable of inhibiting the functional expression or activity of an HDC1 polypeptide.
  • proteins or polypeptides capable of inhibiting the functional expression or activity of an HDC1 polypeptide include e.g. a nucleic acid encoding an antibody (or nanobody etc) that binds to an HDC1 polypeptide and reduces the activity thereof.
  • the binding of the antibody results in increased turnover of the antibody-HDC1 complex by cellular quality control mechanisms.
  • proteins capable of inhibiting the functional expression or activity of an HDC1 polypeptide may also be a dominant negative HDC1 protein or protein fragments.
  • Dominant negative HDC1 proteins could for example be HDC1 proteins wherein HDAC binding sites have been modified, e.g. removed, thereby inhibiting HDAC function.
  • the plant or plant cell can be contacted with molecules interfering with HDC1 function by triggering aggregation of the target protein (interferor peptides) as e.g. described in WO2007/071789 and WO2008/148751 .
  • the plant or plant cell can be contacted with so-called alphabodies specific for HDC1 , i.e. non-natural proteinaceous molecules that can antagonize protein function, as e.g. described in WO2009/030780, WO2010/066740 and WO2012/092970.
  • the reduction of the expression and/or activity of HDC1 is preferably inducible in/by the conditions under which it is desirable to reduce HDC1 expression and/or functions, such as severe stress conditions.
  • inducible expression of the above described nucleic acids expressed in the plant or plant cell that that result in an inhibition of the expression and/or activity of HDC1 in the plant or plant cell is operably linked to an inducible promoter. A list of inducible promoters is described in detail above.
  • HDC1 downregulation can be induced at the desired moment using a spray (systemic application) with inhibitory nucleic acids, such as RNA or DNA molecules that function in RNA-mediated gene silencing (similar to the above described molecules) which target endogenous HDC1 , as e.g. described in WO201 1 /1 12570 (incorporated herein by reference).
  • inhibitory nucleic acids such as RNA or DNA molecules that function in RNA-mediated gene silencing (similar to the above described molecules) which target endogenous HDC1 , as e.g. described in WO201 1 /1 12570 (incorporated herein by reference).
  • the invention provides chimeric genes comprising a nucleic acid which when transcribed results in an increased or decreased activity and/or expression of HDC1 , as described in detail above. Chimeric genes or vectors comprising the chimeric genes are also included in the invention .
  • Nucleic acids and chimeric genes used to practice the invention can be expressed by introduction into a plant cell by any means.
  • nucleic acids or expression constructs can be introduced into the genome of a desired plant host, or, the nucleic acids or chimeric genes can be episomes.
  • Introduction into the genome of a desired plant can also be such that the host's HDC1 protein production is regulated by endogenous transcriptional or translational control elements, or by a heterologous promoter, e.g., a promoter of this invention.
  • "Introducing” in connection with the present application relates to the placing of genetic information in a plant cell or plant by artificial means, such as transformation. This can be effected by any method known in the art for introducing RNA or DNA into plant cells, tissues, protoplasts or whole plants. In addition to artificial introduction as described above, “introducing” also comprises introgressing genes as defined further below.
  • Transformation means introducing a nucleotide sequence into a plant in a manner to cause stable or transient expression of the sequence. Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods can include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium- mediated transformation.
  • PEG polyethylene glycol
  • the invention uses Agrobacterium tumefaciens mediated transformation.
  • Other bacteria capable of transferring nucleic acid molecules into plant cells may be used, such as certain soil bacteria of the order of the Rhizobiales, e.g. Rhizobiaceae (e.g. Rhizobium spp., Sinorhizobium spp., Agrobacterium spp); Phyllobacteriaceae (e.g. Mesorhizobium spp., Phyllobacterium spp.); Brucellaceae (e.g. Ochrobactrum spp.); Bradyrhizobiaceae (e.g.
  • Bradyrhizobium spp. Bradyrhizobium spp.
  • Xanthobacteraceae e.g. Azorhizobium spp.
  • Agrobacterium spp. Rhizobium spp.
  • Sinorhizobium spp. Mesorhizobium spp.
  • Phyllobacterium spp. Ochrobactrum spp.
  • Bradyrhizobium spp. examples of which include Ochrobactrum sp., Rhizobium sp., Mesorhizobium loti, Sinorhizobium meliloti.
  • Rhizobia include R. leguminosarum bv, trifolii, R.
  • leguminosarum bv,phaseoli and Rhizobium leguminosarum, bv, viciae US Patent 7,888,552.
  • Other bacteria that can be employed to carry out the invention which are capable of transforming plants cells and induce the incorporation of foreign DNA into the plant genome are bacteria of the genera Azobacter (aerobic), Closterium (strictly anaerobic), Klebsiella (optionally aerobic), and Rhodospirillum (anaerobic, photosynthetically active).
  • Rhizobiaceae members such as Rhizobium trifolii, Rhizobium leguminosarum and Phyllobacterium myrsinacearum, while Rhizobium sp. NGR234, Sinorhizobium meliloti and Mesorhizobium loti could indeed be modified to mediate gene transfer to a number of diverse plants (Broothaerts et al., 2005, Nature, 433:629-633).
  • making transgenic plants or seeds comprises incorporating sequences used to practice the invention and, in one aspect (optionally), marker genes into a target expression construct (e.g., a plasmid), along with positioning of the promoter and the terminator sequences.
  • a target expression construct e.g., a plasmid
  • This can involve transferring the modified gene into the plant through a suitable method.
  • a construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. For example, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203; Pawlowski (1996) Mol. Biotechnol.
  • protoplasts can be immobilized and injected with a nucleic acids, e.g., an expression construct.
  • a nucleic acids e.g., an expression construct.
  • plant regeneration from protoplasts is not easy with cereals, plant regeneration is possible in legumes using somatic embryogenesis from protoplast derived callus.
  • Organized tissues can be transformed with naked DNA using gene gun technique, where DNA is mated on tungsten microprojectiles, shot 1/100th the size of cells, which carry the DNA deep into cells and organelles. Transformed tissue is then induced to regenerate, usually by somatic embryogenesis. This technique has been successful in several cereal species including maize and rice.
  • a third step can involve selection and regeneration of whole plants capable of transmitting the incorporated target gene to the next generation.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21 -73, CRC Press, Boca Raton, 1985.
  • Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee (1987) Ann. Rev. of Plant Phys. 38:467486. To obtain whole plants from transgenic tissues such as immature embryos, they can be grown under controlled environmental conditions in a series of media containing nutrients and hormones, a process known as tissue culture. Once whole plants are generated and produce seed, evaluation of the progeny begins.
  • Viral transformation may also be used for transient or stable expression of a gene, depending on the nature of the virus genome.
  • the desired genetic material is packaged into a suitable plant virus and the modified virus is allowed to infect the plant.
  • the progeny of the infected plants is virus free and also free of the inserted gene.
  • Suitable methods for viral transformation are described or further detailed e. g. in WO 90/12107, WO 03/052108 or WO 2005/098004.
  • the chimeric gene after the chimeric gene is stably incorporated in transgenic plants, it can be introduced into other plants by sexual crossing or introgression. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. Since transgenic expression of the nucleic acids of the invention leads to phenotypic changes, plants comprising the recombinant nucleic acids of the invention can be sexually crossed with a second plant to obtain a final product. Thus, the seed of the invention can be derived from a cross between two transgenic plants of the invention, or a cross between a plant of the invention and another plant.
  • the desired effects can be enhanced when both parental plants express the polypeptides, e.g., an HDC1 gene of the invention.
  • the desired effects can be passed to future plant generations by standard propagation means.
  • plants are selected using a dominant selectable marker incorporated into the transformation vector.
  • a dominant selectable marker can confer antibiotic or herbicide resistance on the transformed plants, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic or herbicide.
  • modified traits can be any of those traits described above.
  • to confirm that the modified trait is due to changes in expression levels or activity of the transgenic polypeptide or nucleic acid can be determined by analyzing mRNA expression using Northern blots, RT-PCR or microarrays, or protein expression using immunoblots or Western blots or gel shift assays.
  • “Introgressing” means the integration of a gene in a plant's genome by natural means, i.e. by crossing a plant comprising the chimeric gene described herein with a plant not comprising said chimeric gene.
  • the offspring can be selected for those comprising the chimeric gene.
  • the invention further provides plants, plant cells, organs, seeds or tissues that have been modified so as to have an increased expression and/or activity of a protein having the activity of the protein with the amino acid sequence of SEQ ID NO. 6. when compared to a control plant.
  • transgenic plants such as transgenic plants, plant cells, organs, seeds or tissues, comprising and expressing the nucleic acids used to practice this invention resulting in an increased expression and/or activity of an HDC1 polypeptide
  • the invention provides plants, e.g., transgenic plants, plant cells, organs, seeds or tissues that show improved growth under (mild or moderate) stress conditions such as limiting water conditions; thus, the invention provides stress-tolerant, and particularly drought-tolerant plants, plant cells, organs, seeds or tissues (e.g., crops).
  • the invention also provides plants, e.g., transgenic plants, plant cells, organs, seeds or tissues that show improved growth under control conditions; thus, the invention provides plants, plant cells, organs, seeds or tissues (e.g., crops) with increased biomass and/or yield and/or growth rate.
  • the invention further provides plants, e.g., transgenic plants, plant cells, organs, seeds or tissues that show improved growth under limiting water conditions; thus, the invention provides drought-tolerant plants, plant cells, organs, seeds or tissues (e.g., crops).
  • the invention provides plants, e.g., transgenic plants, plant cells, organs, seeds or tissues that show an accelerated flowering time; thus, the invention provides plants, plant cells, organs, seeds or tissues (e.g., crops) with an accelerated flowering time.
  • the invention further provides plants, plant cells, organs, seeds or tissues that have been modified so as to have a reduced expression and/or activity of a protein having the activity of the protein with the amino acid sequence of SEQ ID NO. 6. when compared to a control plant.
  • transgenic plants, plant cells, organs, seeds or tissues comprising and expressing the nucleic acids used to practice this invention resulting in a reduced expression and/or activity of an HDC1 polypeptide
  • the invention provides plants, e.g., transgenic plants, plant cells, organs, seeds or tissues that show enhanced survival under severe stress conditions enhanced recovery after severe stress conditions.
  • plants, e.g., transgenic plants, that show a delayed flowering time are also provided.
  • the reduction in expression and/or activity of a protein having the activity of the protein with the amino acid sequence of SEQ ID NO. 6 is inducible.
  • the plant, plant part, plant organs and plant cell of the invention comprising a nucleic acid used to practice this invention can be dicotyledonous (a dicot) or monocotyledonous (a monocot).
  • Examples of monocots comprising a nucleic acid of this invention, e.g., as monocot transgenic plants of the invention are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (com).
  • dicots comprising a nucleic acid of this invention, e.g., as dicot transgenic plants of the invention, are cotton, tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.
  • dicot transgenic plants of the invention are cotton, tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.
  • plant or plant cell comprising a nucleic acid of this invention include a broad range of plants, including, but not limited to, species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Cojfea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio,
  • the invention furthermore provides propagating material created from the plant of plants cells of the invention.
  • the creation of propagating material relates to any means know in the art to produce further plants, plant parts or seeds and includes inter alia vegetative reproduction methods (e.g. air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, twin- scaling), sexual reproduction (crossing with another plant) and asexual reproduction (e.g. apomixis, somatic hybridization).
  • vegetative reproduction methods e.g. air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, twin- scaling
  • sexual reproduction crossing with another plant
  • asexual reproduction e.g. apomixis, somatic hybridization
  • the plant cell described herein is a non-propagating plant cell or a plant cell that cannot be regenerated into a plant or a plant cell that cannot maintain its life by synthesizing carbohydrate and protein from the inorganics, such as water, carbon dioxide, and inorganic salt, through photosynthesis.
  • a transgenic plant of this invention can also include the machinery necessary for expressing or altering the activity of a polypeptide encoded by an endogenous gene, e.g a gene ecoding a functional HDC1 protein according to the invention, for example, by altering the phosphorylation state of the polypeptide to maintain it in an activated state.
  • Transgenic plants or plant cells, or plant explants, or plant tissues
  • incorporating the nucleic acids of the invention and/or expressing the polypeptides of the invention can be produced by a variety of well-established techniques as described elsewhere in this application.
  • a nucleic acid or polynucleotide can be DNA or RNA, single- or double-stranded. Nucleic acids can be synthesized chemically or produced by biological expression in vitro or even in vivo. Nucleic acids can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • DNA includes cDNA and genomic DNA.
  • protein or "polypeptide” as used herein describe a group of molecules consisting of more than 30 amino acids, whereas the term “peptide” describes molecules consisting of up to 30 amino acids. Proteins and peptides may further form dimers, trimers and higher oligomers, i.e. consisting of more than one (poly)peptide molecule. Protein or peptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc.
  • protein and “peptide” also refer to naturally modified proteins or peptides wherein the modification is effected e.g. by glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.
  • nucleic acid or protein comprising a sequence of nucleotides or amino acids
  • a chimeric gene comprising a nucleic acid which is functionally or structurally defined, may comprise additional DNA regions etc.
  • SEQ ID NO. 1 Promoter region of the Arabidopsis thaliana HDC1 gene
  • SEQ ID NO. 2 overexpression vector pMDC32 35S HDC
  • SEQ ID NO. 3 overexpression vector pUB-DEST Ubi10 HDC1
  • SEQ ID NO. 4 Amino acid sequence Saccharomyces cerevisiae Rxt3 aa
  • SEQ ID NO. 5 Nucleotide sequence of HDC1 from Arabidopsis thaliana
  • SEQ ID NO. 6 Amino acid sequence of HDC1 from Arabidopsis thaliana
  • SEQ ID NO. 7 Nucleotide sequence of HDC1 from Arabidopsis lyrata
  • SEQ ID NO. 8 Amino acid sequence of HDC1 from Arabidopsis lyrata
  • SEQ ID NO. 9 Nucleotide sequence of HDC1 from Populus trichocarpa
  • SEQ ID NO. 10 Amino acid sequence of HDC1 from Populus trichocarpa
  • SEQ ID NO. 1 1 Nucleotide sequence of HDC1 from Medicago truncatula
  • SEQ ID NO. 12 Amino acid sequence of HDC1 from Medicago truncatula
  • SEQ ID NO. 13 Nucleotide sequence of HDC1 from Vitis vinifera
  • SEQ ID NO. 14 Amino acid sequence of HDC1 from Vitis vinifera
  • SEQ ID NO. 15 Nucleotide sequence of HDC1 from Ricinus communis
  • SEQ ID NO. 16 Amino acid sequence of HDC1 from Ricinus communis
  • SEQ ID NO. 17 Nucleotide sequence of HDC1 from Oryza sativa
  • SEQ ID NO. 18 Amino acid sequence of HDC1 from Oryza sativa
  • SEQ ID NO. 19 Nucleotide sequence of HDC1 from Oryza sativa
  • SEQ ID NO. 20 Amino acid sequence of HDC1 from Oryza sativa
  • SEQ ID NO. 21 Nucleotide sequence of HDC1 from Brachypodium distachyon
  • SEQ ID NO. 22 Amino acid sequence of HDC1 from Brachypodium distachyon
  • SEQ ID NO. 24 Amino acid sequence of HDC1 from Sorghum bicolor
  • SEQ ID NO. 25 Nucleotide sequence of HDC1 from Sorghum bicolor
  • SEQ ID NO. 26 Amino acid sequence of HDC1 from Sorghum bicolor
  • SEQ ID NO. 27 Nucleotide sequence of HDC1 from Zea mays
  • SEQ ID NO. 28 Amino acid sequence of HDC1 from Zea mays
  • SEQ ID NO. 29 Nucleotide sequence of HDC1 from Glycine max
  • SEQ ID NO. 30 Amino acid sequence of HDC1 from Glycine max
  • SEQ ID NO. 31 Nucleotide sequence of HDC1 from Glycine max
  • SEQ ID NO. 32 Amino acid sequence of HDC1 from Glycine max
  • SEQ ID NO. 35 Nucleotide sequence of HDC1 from Glycine max
  • SEQ ID NO. 36 Amino acid sequence of HDC1 from Glycine max
  • SEQ ID NO. 37 Nucleotide sequence of HDC1 from Triticum aestivum
  • SEQ ID NO. 38 Amino acid sequence of HDC1 from Triticum aestivum
  • SEQ ID NO. 39 Nucleotide sequence of HDC1 from Solanum lycopersicum
  • SEQ ID NO. 46 HDC1 pairl forward primer (RT-PCR/qPCR)
  • SEQ ID NO. 48 HDC1 pair2 forward primer (RT-PCR/qPCR)
  • SEQ ID NO. 49 HDC1 pair2 reverse primer (RT-PCR/qPCR)
  • SEQ ID NO. 50 HDC1 pair3 forward primer (RT-PCR/qPCR)
  • SEQ ID NO. 51 HDC1 pair3 reverse primer (RT-PCR/qPCR)
  • SEQ ID NO. 52 HDC1 pair4 forward primer (RT-PCR/qPCR)
  • SEQ ID NO. 53 HDC1 pair4 reverse primer (RT-PCR/qPCR)
  • SEQ ID NO. 55 overexpression vector pTVE704 Examples
  • Ubi10::HDC1 in pUB-Dest 35S::GFP-HDC1 in pH7WGF2 (Karimi et al., 2002, Trends Plant Sci 7:193-195), Ubi10::GFP-HDC1 pUBN-GFPDest (Grefen et al., 2010, Plant J 64:355-365), 35S::nYFP-HDC1/cYFP- HDA6/HDA19/SIN3 in pBiFCt-2in1 -NN, 35S::nYFP-SIN3/cYFP-HDA19 in pBiFCt-2in1 -NN (Grefen and Blatt, 2012, Biotechniques 53:31 1-314).
  • HDC1 antibody was raised in rabbit (Agrisera) using a synthetic peptide matching amino acids 341 -356 in the HDC1 sequence, and affinity purified. An extra cysteine was added to the N-terminus to improve binding capacity.
  • H3K9/K14Ac and H3 antibodies were purchased from Diagenode (pAb-005-044) and Abeam (ab1791 ). His-tag antibody was obtained from NEB (#2366).
  • Plasmids were inserted by heat shock into Agrobacterium tumefaciens strain GV3101 pMP90 (Koncz and Schell, 1986, Mol. Gen. Genet. 204: 383-396).
  • Agrobacterium-mediated transformation of . thaliana was performed by the floral-dip method (Clough and Bent, 1998, Plant J. 16, 735-743). Homozygous T 2 progenies were used for germination tests.
  • Agrobacterium-mediated transient transformation of N. tabacum and N. benthamiana was achieved by leaf infiltration (Geelen et al., 2002, Plant Cell 14: 387-406).
  • Chromatin extraction and immunoprecipitation were carried out following published protocols ((Gendrel et al., 2002, Science 297, 1871 -1873; Saleh et al., 2008, Plant Cell 20, 568-579).
  • tissue samples were incubated in 1 % (w/v) formaldehyde for 15 min under vacuum.
  • Cross-linking was stopped by adding 125 mM glycine, and tissues were rinsed, blotted dry and frozen.
  • Diluted chromatin extracts were incubated with antibody against H3K9/K14Ac (Diagenode pAb-005-044) following the manufacture instructions.
  • IP-DNA Immunoprecipitated chromatin-DNA
  • input chromatin-DNA was reverse cross-linked and residual protein was removed by proteinase K treatment.
  • DNA was recovered by phenol/chloroform extraction and ethanol precipitation. DNA then was re-suspended and purified by MinElute Reaction Cleanup kit (QIAGEN).
  • MinElute Reaction Cleanup kit QIAGEN.
  • DNA samples were amplified using GenomePlex Complete Whole Genome Amplification (WGA2, Sigma-Aldrich) following the manufacturer's protocol.
  • GST- or His-tagged proteins were expressed in E. coli BL21 cells. Following induction with 1 mM IPTG cells were harvested and sonicated in lysis buffer. The soluble HDC1 -His, GST-HDA6 and GST-HDA19 proteins were affinity- purified using the Ni-NTA (Sigma) and Glutathione-Sepharose resin (GE Healthcare) according to the manufacturer' instructions. For pull-down assays, GST-tagged proteins were bound to Glutatione-Sepharose resin and applied to a microcolumn.
  • HDC1 -His or nuclei-enriched plant lysates were combined with 1X protein inhibitor (Complete Mini, 1 1836153001 , Roche, UK) in Tris-NaCI buffer. Samples were incubated overnight on ice. After several washes, pulled down protein was eluted in 1X Laemmli Buffer.
  • YFP fluorescence was excited at 514 nm with light from Argon laser and was collected using lambda mode between 520-550 nm.
  • Co-localization plane and line scans were evaluated using Zeiss LSM 510 AIM software (v3..2).
  • ABA1 (ABA DEFICIENT 1 ): AT5G67030; ABA3(ABA DEFICIENT 3): AT1 G16540; ABI3 (ABA INSENSITIVE 3): AT3G24650; AFP3 (ABI FIVE BINDING PROTEIN) 3: AT3G29575; DR4 (DROUGHT-REPRESSED 4): AT1 G73330; FLC (FLOWERING LOCUS C): AT5G10140; FUS3 (FUSCA3): AT3G26790; HDC1 (H ISTONE DEACETYLATION COMPLEX 1 ): AT5G08450; HDA6 (HISTONE DEACETYLASE 6): AT5G631 10; HDA19 (H ISTONE DEACETYLASE 19): AT4G38130; LEC1 (LEAFY COTYLEDON 1 ): AT1 G21970; PYL4 (PYR1 -LIKE 4): AT2G38310; RAB18
  • HDC1 is a non-redundant, ubiquitous, nuclear protein
  • HDC1 (At5g08450) is a single-ropy gene in A. thaliana. Predicted splice variants only differ in the upstream UTR. Unique HDC1 homologues are also present in all other plant species for which genome information is currently available, including important crops such as maize and rice (Fig. 1A).
  • the -900 amino-acid long sequence of the predicted plant HDC1 proteins contains a -300 amino-acid long sequence in the C-terminal half that is highly similar to Rxt3 proteins, which are ubiquitously present in lower eukaryotes but remain functionally uncharacterized (alignment in Fig. 1 C).
  • GFP green fluorescent protein
  • Example 3 HDC1 physically interacts with HDA6 and HDA19 and promotes histone deacetylation
  • HDC1 is a member of HDAC protein complexes in plants.
  • Direct interaction was investigated by bimolecular fluorescence complementation (BiFC).
  • HDC1 c Expression of the genomic sequence of HDC1 under its own promoter in the hdd -1 background (HDC1 c) reverted this phenotype; H3K9K14ac:H3 in the complementation line was similar to wildtype (Fig. 5C).
  • HDC1 interacts with histone deacetylases and is required for histone deacetylase activity in planta.
  • HDC1 transcript levels in the other T-DNA insertion lines were similar to those in wildtype or even higher Fig. 7A,B).
  • HDC1 c complementation lines were obtained by expressing genomic HDC1 under its own promoter (646 bp upstream sequence) in hdd -1 background.
  • TSA histone deacetylase inhibitor trichostatin A
  • TSA increased the ABA-sensitivity of wildtype plants in a dose-dependent manner, with 0.3 ⁇ producing a significant effect at 0.2 ⁇ ABA and 3 ⁇ TSA producing a significant effect at 0.4 ⁇ ABA. Furthermore, addition of TSA increased ABA-sensitivity of the HDC1 -overexpressing lines. Thus ABA-sensitivity of germinating seeds and de- sensitization of seedlings towards ABA by HDC1 -overexpression depend on the catalytic activity of histone deacetylases.
  • Example 6 HDC1 does not impact on vegetative development but is required for flowering
  • HDAC mutants Several developmental phenotypes have been reported for HDAC mutants. For example, hda6/hda19 double mutants display embryonic structures on mature leaves and do not repress embryo-specific transcription factors such as LEC1 , FUS3 and ABI3 after germination (Tanaka et al., 2008, supra). By contrast, leaves of hdd -1 plants were normal and LEC1 and FUS3 were effectively repressed already two days after germination (DAG, Fig.9). ABI3 transcript was still present at 2 DAG, with hdc1 -1 plants expressing higher levels and HDC1 -OX plants expressing lower levels than wildtype plants, but was reduced to very low levels in all lines by 6 DAG. We conclude that in control conditions HDC1 is not required for successful progression of seedlings into the vegetative growth phase.
  • HDA6-overexpressing plants had similar weights as wildtype plants (Fig. 10) and therefore did not phenocopy HDC1 -overexpressing lines.
  • Example 8 HDC1 alters transcript levels and acetylation status of salt stress-regulated genes.
  • transcript levels of the genes were similarly low in all lines apart from ABA1 transcript which was increased in hdd -1 .
  • Shoot ABA levels confirmed that ABA biosynthesis was efficiently induced by salt in al lines but attained levels were slightly higher/lower in hdc1 -1/OX lines (Fig. 17).
  • Acetylation status of other genes remain to be tested.
  • the results identify ABA1 , RD29B, PYL4 and DR4 as direct targets of HDC1 -facilitated histone de-acetylation, and they provide a mechanistic explanation for the altered transcriptional responses of these genes in the mutants.
  • Example 9 The growth-enhancing effect of HDC1 overexpression is maintained under water stress
  • HDC1 -OX lines raised our curiosity about the net outcome of these potentially counter-productive features on plant performance under water or salt stress.
  • HDC1 mutant lines and wildtype plants were subjected to a controlled water-limiting regime in short- day conditions that started on day 14 and imposed a continuous relative soil water content of -50% of the control condition for the remainder of the experiment (Fig .19A). Differences in growth between the lines were apparent in larger (HDC1 -OX) and smaller (hd1 -1 ) rosette diameters of younger plants, recorded on day 14 and 28.
  • the 2757 bp coding sequence of the A. thaliana HDC1 gene (SEQ ID NO.: 5) was optimized for wheat codon usage (resulting in the nucleotide sequence of SEQ ID NO: 54).
  • a Bsal site was created at the ATG and a Mlul site behind the stop codon.
  • a gel-purified Bsal-Mlul fragment containing the optimized hdd gene was ligated between the maize ubiquitin-1 promoter PubiZm and a nos terminator in a Ncol-Mlul digested vector pTCD145 that contains in addition a P35S:bar selectable marker cassette.
  • the ligation reaction product was used to transform MC1061 bacterial cells. Antibiotic marker-resistant colonies were isolated and verified by restriction digest analysis and sequencing.
  • the plant transformation vector pTVE704 used for the generation of the wheat transgenics contains two expression cassettes.
  • the selectable marker cassette has the 35S promoter driving the Bar gene and the hdd cassette has the maize ubiquitin-1 promoter driving the codon optimized A. thaliana HDC1 coding sequence.
  • the pTVE704 vector backbone is derived from pGSC1700 (Cornelissen and Vandewiele, 1989: Nuclear transcriptional activity of the tobacco plastid psbA promoter. Nucleic Acids Research, 17, 19-25).
  • Plasmids were inserted by heat shock into Agrobacterium tumefaciens strain AGL1 (Lazo et al. 1991 ).
  • Agrobacterium-mediated transformation of Triticum aestivum immature embryos was performed using a modification of the Rothamsted method (Wu et al. 2003: Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Reports, 21, 659-668). Plants were selected using media containing PPT and regenerated plantlets were transferred to the greenhouse to obtain multiple events. Single copy events were confirmed by Southern Blot analysis.
  • Example 11 Effect of HDC1 overexpression in wheat on biomass
  • HDC1 overexpression constructs are transformed into crop plants other than wheat according to standard methods known in the art and overexpression is confirmed by RT-PCR, Northern or western blotting. Biomass (of vegetative tissue and seeds) of plants overexpressing HDC1 grown under various stress conditions as described above (e.g. water limiting conditions, salt stress, osmotic stress) or grown under non-stress condition are compared to wt plants grown under the same conditions. An increased biomass is observed in HDC1 -overexpression plants compared to wt, both under stress and under non-stress conditions.
  • stress conditions e.g. water limiting conditions, salt stress, osmotic stress
  • HDC1 seeds of the above plants overexpressing HDC1 are subjected to ABA, osmotic stress and/or histone deacetylase inhibitors, and germination was compared to seeds of control plants as described above. Germination of the HDC1 overexpressing seeds was less inhibited by the above treatment compared to wt seeds.
  • flowering time, seed yield and plant height of HDC1 -overexpressing crop plants is compared to that of wt plants. Overexpressing plants display an earlier flowering time than wt plants, an increased seed yield and increased plant height as compared to wt plants.

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Abstract

L'invention concerne des procédés de production d'une plante ayant une meilleure tolérance aux contraintes et un meilleur rendement, ainsi que des gènes chimères utilisés selon ces procédés et une plante comprenant ces gènes chimères.
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CN104955947A (zh) 2015-09-30
CA2899274A1 (fr) 2014-08-07
BR112015017830A2 (pt) 2017-11-21
AU2014211570A1 (en) 2015-07-23
WO2014118123A1 (fr) 2014-08-07

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